Hemostasis can be defined as an essential homeostatic system leading to the formation of a hemostatic clot. Hemostatis has to be finely tuned in order to avoid the risk of bleeding, or haemorrhage, and the appearance of pathologic obstructive blood clots, also referred herein as thrombosis. Acute thrombotic events remain the main cause of mortality and morbidity in western countries.
The formation of a clot results from the coordinated activation of the coagulation cascade and of platelets. This is an amplified process with activating platelets supporting the assembly of coagulation enzymatic complexes and the final enzyme of the coagulation, thrombin being a potent activator of platelets. The final product is composed of aggregates of platelets expressing specific sites at their surface and of polymerized fibrin fibres. The respective role of coagulation and platelets may vary according to the vascular bed and rheological conditions, platelets and fibrin being more predominant in arterial and venous thrombi respectively.
For a better comprehension of coagulation as a dynamic process, the man skilled in the art may also refer for complementary information to Kottke-Marchant & Lefkowitz (KOTTKE-MARCHANT & LEFKOWITZ, 2008, Chapter 1—Coagulation Pathway and Physiology. An Algorithmic Approach to Hemostasis Testing).
When a hemostatic clot is formed, one has to consider additional biological mechanisms that provoke secondary clot resorbtion, allowing tissue reparation (healing). Those secondary mechanisms may be commonly referred as the clot removal system, or «fibrinolysis».
One has to consider that a clot is in permanent equilibrium between building and destruction. When a thrombotic clot is formed in an artery of vein, spontaneous resorbtion of the clot is too delayed to be efficient to protect downstream tissue from irreversible ischemia. Thus two strategies are currently used to treat thrombosis: (i) to limit the incremental formation of the clot by antiplatelet and anticoagulant drugs and (ii), to enhance clot destruction by inducing fibrinolysis.
The invention refers to this second strategy.
The fibrinolytic pathway is composed of both activators and inhibitors. Thus, an «activator» of the fibrinolytic pathway may have the ability to provoke lysis of fibrin, whereas an «inhibitor» of the fibrinolytic pathway may have the opposite effect.
During its formation, the clot exposes the elements required for its resorbtion: fibrin exposes binding sites for the circulating zymogen plasminogen and its activator, the tissue-type plasminogen activator (t-PA). The formation of a ternary complex in which fibrin, plasminogen and t-PA are associated, ensure the efficacy of the fibrinolysis and prevents its extension to the blood stream (fibrinogenolysis). Fibrin-bound plasminogen is cleaved at Arg-561-Val562 by its activator t-PA, generating the disulfide bond linked two chain protease plasmin.
It is well known that t-PA and plasmin bind to cationic residues, mainly amine (NH2) groups of lysine residues expressed on the fibrin network (Lijnen et al., 2001, Elements of the fibrinolytic system. Ann N Y Acad Sci, 936, 226-236).
Furthermore, plasminogen binds with a high affinity to carboxy terminal lysines (Lys). Plasmin cleavage of fibrin exposes new carboxy-terminal Lys, leading to more plasminogen binding sites and more plasmin formed (amplification process). In contrast, TAFI (Thrombin-Activable Fibrinolysis Inhibitor), a carboxypeptidase, removes the carboxy terminal Lys, preventing binding of plasminogen and inhibiting fibrinolysis. The t-PA also binds to side chain free amine.
Lysine mimetics, such as □-amino caproic acid or tranexamic acid (Royston, 1995, Blood-sparing drugs: Aprotinin, tranexamic acid, and epsilon-aminocaproic acid, Int Anesthesiol Clin), displace plasminogen from C-terminal lysine residues, thus limiting fibrinolysis. On the other hand, amine-bound t-PA is protected from inhibition by serpins, mainly the plasminogen activator inhibitor (PAI-1) also known as endothelial plasminogen activator inhibitor or as serpin E1.
Acute non-interventional treatment remains mainly the intravenous injection of recombinant tissue plasminogen activator, also referred herein as rt-PA (Altéplase, Actilyse® or Tenecteplase, Metalyse®, Boehringer Ingelheim). For example international guidelines recommend the earliest IV injection of recombinant t-PA in acute stroke (group IST et al., 2012, The benefits and harms of intravenous thrombolysis with recombinant tissue plasminogen activator within 6 h of acute ischaemic stroke (the third international stroke trial [ist-3]): A randomised controlled trial, Lancet). But the efficacy of recombinant t-PA peripheral injection is limited to 10% due to:    dilution of the potent compound in the whole blood,    inhibition of recombinant t-PA by circulating chelators such as PAI-1 during its plasma traffic,    low initial level of rt-PA binding to the thrombus.    increased risk of hemorrhage for high doses and delayed injection.
Furthermore, although thrombolytic effects of t-PA are beneficial, its neurotoxicity, at the required high dose ranges that are presently used, is problematic.
To tentatively protect rt-PA from plasma inhibitors, it is conditioned in the presence of Arg (Alteplase formulation). The protective effect of amine residues has also been exploited with the use of annexin 2 as a chaperone. Indeed, the colocalisation of plasminogen and t-PA at the cell surface allows cells to play a regulatory role on fibrinolysis. The annexin2-S100A10 complex provides such a platform for the activation of plasminogen thanks to the presentation of a C-terminal Lys in the correct three dimensional orientation for recognition of t-PA and plasminogen (Madureira et al., 2011 The role of the annexin A2 heterotetramer in vascular fibrinolysis, Blood), In this context, annexin A2 has been proposed with success, as a chaperone for IV injection of t-PA (Zhu et al., 2010, Annexin 2 combined with low-dose t-PA improves thrombolytic therapy in a rat model of focal embolic stroke, J Cereb Blood Flow Metab). Limitations to this strategy are the absence of t-PA vectorization and the fact that annexin A2 is a recombinant protein highly expensive to produce.
There is a general need for improved treatments related to acute vascular thrombotic diseases. There is thus an urgent need for new forms of t-PA, or t-PA-derived active compounds more therapeutically effective at amounts of t-PA lower than those currently administered, so as to at least temperate the toxic side effects of this active ingredient.
Thus, protection of t-PA during its plasma circulation and its efficient vectorization to the thrombus represent an important challenge.